Tissue-type plasminogen activator (t-PA) is a serine protease consisting of several domains, which catalyzes the conversion of plasminogen to plasmin and is employed for fibrinolytic therapy.
A large number of t-PA variants and mutations are known, as will be seen for instance, by review of articles by T. J. R. Harris. Prof. Eng. 1: 449-459 (1987) and J. Krause, Fibrinolysis 2: 133-142 (1988).
It is known, inter alia, that fibrinolysis is regulated partly by the interaction between t-PA and the plasminogen activator inhibitor 1 (PAI-1), a serine protease inhibitor from the serpine family. The binding of PAI-1 to t-PA is essentially accomplished via amino acids 296-302 of t-PA. Mutation of this region causes a reduction of the inhibitory influence of PAI-1 on t-PA (E. L. Madison et al. (1990)). Extensive investigations have been carried out on the mechanism of the interaction between amino acid region 296-302 of t-PA and PAI-1 (cf. E. L. Madison, Nature 339 (1989) 721-723, R. V. Schohet, Thrombosis and Haemostasis 71 (1994) 124-128, C. J. Refino, Thrombosis and Haemostasis 70 (1993) 313-319, N. F. Paoni, Protein Engineering 6 (1993) 529-534 and Thrombosis and Haemostasis 70 (1993) 307-312, W. F. Bennett, J. Biol. Chem. 266 (1991) 5191-5201, D. Eastman, Biochemistry 31 (1992) 419-422.
Unmodified t-PA, in its form as occurs in plasma (i.e., "wild type" t-PA), consists of 527 amino acids, and can be split by plasmin into two chains which are then still held together via a disulfide bridge. The A chain (also referred to as the heavy chain) consists of four structural domains. The finger domain (amino acids 1-49) displays certain similarities to the finger structures in fibronectin. The growth factor domain (amino acids 50-86), is to a certain extent, homologous to murine and human epidermal growth factors. The two kringle domains (amino acids 87-175 and 176-262) are to a large extent homologous to the fourth and fifth kringle domain of plasminogen. The finger domains and the kringle 2 domains of t-PA are especially involved in fibrin binding and in the stimulation of proteolytic activity by fibrin. The B chain of t-PA (amino acids 276-527, protease domain) is a serine protease and is largely homologous to the B chains of urokinase and plasmin (T. J. R. Harris, supra and J. Krause, supra). t-PA variants which exhibit lower bleeding side effects are described in WO 93/24635 and by B. A. Keyt et al., PNAS USA 91: 3670-3674 (1994). These t-PA variants have an additional glycosylation site at amino acid positions 103-105. In addition, these t-PA variants may be modified at amino acids 296-302, whereby fibrin specificity is increased.
The object of the present invention is to provide thrombolytically active proteins which, have, inter alia lower bleeding side effects as compared to the known plasminogen activators.